UNIT 10.

Preparation of Genomic DNA from Mammalian Tissue

Tissue is rapidly frozen and crushed to produce readily digestible pieces. The processed tissue is placed in a solution of proteinase K and sodium dodecyl sulfate and incubated until most of the cellular protein is degraded. The digest is deproteinized by successive phenol/chloroform/isoamyl alcohol extractions, recovered by ethanol precipitation, and dried and resuspended in buffer. Materials Liquid nitrogen Digestion buffer Phosphate-buffered saline (PBS; APPENDIX 2), ice-cold 25:24:1 phenol/chloroform/isoamyl alcohol (UNIT 10.1) 7.5 M ammonium acetate 100% and 70% ethanol TE buffer, pH 8 (APPENDIX 2) Sorvall H1000B rotor (or equivalent) Beginning with whole tissue: 1a. As soon as possible after excision quickly mince tissue and freeze in liquid nitrogen.
If working with liver, remove the gallbladder, which contains high levels of degradative enzymes.

BASIC PROTOCOL

2a. Starting with between 200 mg and 1 g, grind tissue with a prechilled mortar and pestle, or crush with a hammer to a fine powder (keep the tissue fragments). 3a. Suspend the powdered tissue in 1.2 ml digestion buffer per 100 mg tissue. There should be no clumps. Beginning with tissue culture cells: 1b. Spin suspension cultures out of their serum-containing media. Trypsinize adherent cells and collect cells from the flask. Centrifuge 5 min at 500 g (1200 rpm in Sorvall H1000B rotor) and discard supernatant.
A buffered salt solution containing 0.5% (w/v) trypsin and 0.2% (w/v) EDTA is used to dissociate adherent cells from tissue culture dishes (trypsinization). This solution can be purchased.

2b. Resuspend cells with 1 to 10 ml ice-cold PBS. Centrifuge 5 min at 500 g and discard supernatant. Repeat this resuspension and centrifugation step. 3b. Resuspend cells in 1 vol digestion buffer. For <3 107 cells use 0.3 ml digestion buffer. For larger numbers of cells use 1 ml digestion buffer/108 cells. 4. Incubate the samples 12 to 18 hr with gentle shaking at 50C in tightly capped tubes.
The samples will be viscous. After 12 hr incubation the tissue should be almost indiscernible, a sludge should be apparent from the organ samples, and tissue culture cells should be relatively clear.

5. Thoroughly extract the samples with an equal volume of phenol/chloroform/ isoamyl alcohol (UNIT 10.1).
CAUTION: Phenol is extremely caustic.
Preparation of Genomic DNA from Mammalian Tissue

6. Centrifuge 10 min at 1700 g (3000 rpm in Sorvall H1000B rotor).

If the phases do not resolve well, add another volume of digestion buffer, omitting

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Supplement 8 CPI Copyright 1993 by Current Protocols

proteinase K, and repeat the centrifugation. If there is a thick layer of white material at the interface between the phases, repeat the organic extraction.

7. Transfer the aqueous (top) layer to a new tube and add 12 vol of 7.5 M ammonium acetate and 2 vol (original) of 100% ethanol. The DNA should immediately form a stringy precipitate. Recover DNA by centrifuging 2 min at 1700 g.
This brief precipitation in the presence of high salt reduces the amount of RNA in the DNA. For long-term storage it is convenient to leave the DNA in the presence of ethanol. Alternatively, to prevent shearing of high-molecular-weight DNA, omit steps 7 to 9 and remove organic solvents and salt from the DNA by at least two dialysis steps against at least 100 vol TE buffer (see APPENDIX 3). Because of the high viscosity of the DNA, it is necessary to dialyze for a total of at least 24 hr.

8. Rinse the pellet with 70% ethanol. Decant ethanol and air dry the pellet.
It is important to rinse well to remove residual salt and phenol.

9. Resuspend DNA in TE buffer until dissolved. DNA may be shaken gently at room temperature or at 65C for several hours to facilitate solubilization.
If necessary, residual RNA can be removed at this step by adding 0.1% sodium dodecyl sulfate (SDS) and 1 g/ml DNase-free RNase and incubating 1 hr at 37C, followed by organic extraction and ethanol precipitation, as above.

10. Store at 4C; 1 mg/ml DNA is a convenient working concentration. From 1 g mammalian cells, 2 mg DNA can be expected. REAGENTS AND SOLUTIONS Digestion buffer 100 mM NaCl 10 mM TrisCl, pH 8 25 mM EDTA, pH 8 0.5% SDS 0.1 mg/ml proteinase K
The proteinase K is labile and must be added fresh with each use.

COMMENTARY Background Information

There are a number of different procedures for the preparation of genomic DNA. They all start with some form of cell lysis, followed by deproteinization and recovery of DNA. The main differences between various approaches lie in the extent of deproteinization and in molecular weight of the DNA produced. The isolation procedure described here is relatively brief and relies on the powerful proteolytic activity of proteinase K combined with the denaturing ability of the ionic detergent SDS. Use of proteinase K for DNA purification was described by Gross-Bellard et al. (1972) and Enrietto et al. (1983). EDTA is included in the digestion buffer to inhibit DNases.

Critical Parameters
To minimize the activity of endogenous nucleases, it is essential to rapidly isolate, mince, and freeze tissue. Tissue culture cells should be cooled and washed quickly. As soon as the tissue is frozen or the tissue culture cells are added to the lysis buffer, DNA is protected from the action of nucleases throughout this protocol. It is important that the tissue be well dispersed rather than left in large lumps to permit rapid and efficient access to proteinase K and SDS. It is crucial to generate very high-molecular-weight DNA for construction of phage (>60 kb) or cosmid (>120 kb) genomic libraries. Two main precautions should be taken to maximize

Molecular Biology

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Current Protocols in Immunology Supplement 2

molecular weight: (1) minimize shearing forces by gentle (but thorough) mixing during extraction steps, and (2) after the extraction, remove organic solvents and salt from the DNA by dialysis, rather than by ethanol precipitation. The absence of both cellular proteins and proteinase K in the final DNA solution is important for susceptibility of the genomic DNA to restriction enzyme action; therefore, care should be exercised in deproteination. To remove protein completely it may be necessary to repeat the proteinase K digestion. In general, highly pure DNA has an A260/A280 ratio >1.8, while 50% protein/50% DNA mixtures have A260/A280 ratios of 1.5.

Anticipated Results
Approximately 2 mg DNA should be obtained from 1 g tissue or 109 cells. The DNA should be at least 100 kb long and should be digestible with restriction enzymes.

Time Considerations
This protocol involves effort on 2 days: tissue preparation on the first day followed by overnight lysis, and extraction/precipitation on the second day. Actual time spent on the procedure, however, will be less than 1 hr each day. The DNA can be stored indefinitely in the presence of ethanol at 20C.

Literature Cited Troubleshooting

Failure of the organic phase to separate cleanly from the aqueous phase is generally due to a very high concentration of DNA and/or cellular debris in the aqueous phase. Dilution with more digestion buffer and reextraction can remedy this problem. Upon addition of the room-temperature ethanol to the extracted DNA solution, the DNA should precipitate in long, stringy fibers. If there is no precipitate or if the precipitate is flocculent, the DNA is either degraded or not purified away from cellular debris. Improper handling of the tissue before digestion or too much tissue in the digestion reaction are possible causes of such problems.
Enrietto, P.J., Payne, L.N., and Hayman, M.J. 1983. A recovered avian myelocytomatosis virus that induces lymphomas in chickens. Pathogenic properties and their molecular basis. Cell 35:369-379. Gross-Bellard, M., Oudet, P., and Chambon, P. 1973. Isolation of high-molecular-weight DNA from mammalian cells. Eur. J. Biochem. 36:3238.

Contributed by William M. Strauss Whitehead Institute Cambridge, Massachusetts

Preparation of Genomic DNA from Mammalian Tissue

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Supplement 2 Current Protocols in Immunology